The present invention relates to methods for preparing collagen from tissues of humans and other animals. In particular, the present invention provides methods for the preparation of collagen suitable for biomedical, veterinary, and other applications.
Collagen is the most abundant protein in mammals. (See, U.S. Pat. No. 5,043,426 to Goldstein, herein incorporated by reference). Indeed, it represents 30% of the dry weight of the human body. (See, L. C. Junqueira and J. Carneiro, Basic Histology, 4th ed., Lange Medical Publications, Los Altos, Calif. [1983], pp. 89-119). Vertebrate collagen is actually a family of proteins produced by several cell types. Within this protein family, the collagen types are distinguishable by their chemical compositions, different morphological and pathological features distributions within tissues, and their functions. Although many types of collagen have been described, five major types have been recognized.
A. Forms of Collagen
Collagen type I is the most abundant form of collagen, with widespread distribution within the body. It is present in tissues in structures classically referred to as xe2x80x9ccollagen fibersxe2x80x9d that form bones, dentin, tendons, fascias, sclera, organ capsules, dermis, fibrous cartilage, etc. The primary function of type I collagen is to resist tension. Microscopically, type I collagen appears as closely packed, thick. non-argyrophilic, strongly birefringent red or yellow fibers. Its ultrastructure is characterized as being densely packed, thick fibrils with marked variation in diameter. It is produced by fibroblasts, osteoblasts, odontoblasts, and chondroblasts.
Collagen type II is primarily found in cartilage (e.g., hyaline and elastic cartilages). The primary function of type II collagen is to resist intermittent pressure Microscopically, it appears as a loose, collagenous network, that is visible only with picrosirius stain and polarization microscopy. Ultrastructurally, it is characterized as appearing to have no fibers, but with very thin fibrils embedded in abundant ground substance. It is produced by chondroblasts.
Collagen type III is commonly associated with type I collagen in tissues, and may be the collagenous component of reticular fibers. It is present in smooth muscles, endoneurium, arteries, uterus, liver, spleen, kidney, an lung tissue. The primary function of type III collagen is to maintain the structure of expansible organs. Microscopically, it appears as a loose network of thin, argyrophilic, and weakly birefringent greenish fibers. Ultrastructurally, it is characterized as being loosely packed thin fibrils with fairly uniform diameters. It is produced by smooth muscle fibroblasts, reticular cells, Schwann cells, and hepatocytes.
Collagen type IV is found in the epithelial and endothelial basal lamina and basement membranes. The primary function of type IV collagen involves support and filtration. Microscopically, it appears as a thin, amorphous, weakly birefringent membrane. Ultrastructurally, it appears to have neither fibers nor fibrils.
Collagen type V is found in fetal membranes, blood vessels, placental basement membrane, and in small amounts in other tissues. This type of collagen remains largely uncharacterized.
B. Structure Of Collagen
The principal amino acids found in collagen are glycine, proline and hydroxyproline. Hydroxylysine is also characteristic of collagen. These hydroxy amino acids are the result of hydroxylation of proline and lysine present in nascent collagen polypeptides during collagen synthesis. The collagen content in a tissue can be determined by measurement of its hydroxyproline content.
Collagen is comprised of polypeptide chains, designated as xe2x80x9cxcex1.xe2x80x9d There are two types of a chains, referred to as xe2x80x9calpha-1xe2x80x9d (xe2x80x9cxcex11xe2x80x9d) and xe2x80x9calpha-2 (xe2x80x9caxe2x88x922xe2x80x9d). The most important types of xcex11 chains are xcex11(I), xcex11(II), xcex11(III), and xcex11(IV), which aggregate in different combinations to produce the triple helices of types I, II, III, IV, and V. Type I collagen is composed of two xcex11 and one xcex12 chains. It""s formula is (xcex11[I])2, xcex12. The formula for type II collagen is (xcex11[II])4, while the formula for type III collagen is (xcex11[III])3, and type IV is (xcex11[IV])3.
xe2x80x9cTropocollagenxe2x80x9d is the protein unit that polymerizes into aggregations of microfibrillar subunits packed together to form xe2x80x9ccollagen fibrils.xe2x80x9d Hydrogen bonds and hydrophobic interactions are critical in this aggregation and packing. Covalent crosslinks reinforce the structure of the collagen fibrils. Collagen fibrils are thin and elongated, of variable diameter, and have transverse striations with a characteristic periodicity of 64 nm. The transverse striations is produced by the overlapping organization of the subunit tropocollagen molecules. In type I and III collagen, these fibrils associate to produce collagen xe2x80x9cfibers.xe2x80x9d In collagen type I, collagen xe2x80x9cbundlesxe2x80x9d may be formed by association of the fibers. Collagen type II is observed as fibrils, but does not form fibers, while types IV and V do not form fibrils or fibers.
Collagen fibers are the most abundant fiber found in connective tissue. Their inelasticity and molecular configuration provide collagen fibers with a tensile strength that is greater than steel. Thus, collagen provides a combination of flexibility and strength to the tissues in which it resides. In many parts of the body, collagen fibers are organized in parallel arrays to form collagen xe2x80x9cbundles.xe2x80x9d
When fresh, collagen fibers appear as colorless strands, although when a large number of fibers are present, they cause the tissues in which they reside to be white (e.g., tendons and aponeuroses). The organization of the elongated tropocolliagen in the fibers cause them to be birefringent. Staining with certain acidic dyes (e.g., Sirius red) enhances this birefringency. As this increase in birefringency us only observed in oriented collagen structures, it is useful as a method to detect the presence of collagen in a tissue.
C. Properties and Uses of Collagen
There are many properties of collagen that make it an attractive substance for various medical applications, such as implants, transplants, organ replacement, tissue equivalents, vitreous replacements, plastic and cosmetic surgery, surgical suture, surgical dressings for wounds, burns, etc. (See e.g., U.S. Pat. Nos. 5,106,949, 5,104,660, 5,081,106, 5,383,930, 4,485,095, 4,485,097, 4,539,716, 4,546,500, 4,409,332, 4,604,346, 4,835,102, 4,837,379, 3,800,792, 3,491,760, 3,113,568, 3,471,598, 2,202,566, and 3,157,524, all of which are incorporated herein by reference; J. F. Prudden, Arch. Surg. 89:1046-1059 [1964]; and E. E. Peacock et al. Ann. Surg., 161:238-247 [1965]). For example, by itself, collagen is a relatively weak immunogen, at least partially due to masking of potential antigenic determinants within the collagen structure. Also, it is resistant to proteolysis due to its helical structure. In addition, it is a natural substance for cell adhesion and the major tensile load-bearing component of the musculoskeletal system. Thus, extensive efforts have been devoted to the production of collagen fibers and membranes suitable for use in medical, as well as veterinary applications.
Collagen has been used in the area of soft tissue augmentation, as a replacement for paraffin, petrolatum, vegetable oils, lanolin, bees wax, and silicone previously used. (See e.g., U.S. Pat. No. 5,002,071, herein incorporated by reference). However, problems have been associated with the use of collagen in implants. As the non-collagenous proteins present in impure collagen preparations are more potent immunogens than the collagen, and can stimulate the inflammatory response, it is critical that highly pure collagen be used. If the inflammatory cascade is stimulated, the resorption of collagen occurs by the infiltrating inflammatory cells (e.g., macrophages, and granulocytes) that contain collagenase, resulting in thee digestion of the collagen. In addition, collagen itself is chemotactic, and becomes increasingly chemotactic as it is degraded into smaller peptide fragments. Also, there are concerns associated with the use of non-human collagen. For example, a repeatedly documented problem associated with the use of bovine collagen as a biomaterial is the consistent, chronic cellular inflammatory reaction that is evident following its implantation or use. This inflammation may result in residual scar tissue formation, adhesion formation, interference with healing of skin edges, pseudointima formation, pseudodiaphragm formation, disruption of anastomoses, transient low grade fever, aneurysms, or other problems.
D. Preparation of Collagen
Collagen preparations are typically prepared from skin, tendons (e.g., bovine Achilles, tail, and extensor tendons), hide or other animal parts, by procedures involving acid and/or enzyme extraction. Basically, collagen preparation methods involve purification of collagen by extraction with diluted organic acids, precipitation with salts, optional gelation and/or lyophilization, tangential filtration etc. After separating facia, fat and the impurities, the tissue is subjected to moderate digestion with proteolytic enzymes, such as pepsin, then the collagen is precipitated at a neutral pH, redissolved and the residual impurities precipitated at an acid pH. The tissue is then digested with a strong alkali and then exposed to acid to facilitated swelling. The collagen fibers are then precipitated with salts or organic solvents, and dehydrating the collagen fibers. (See e.g., U.S. Pat. No. 5,028,695, herein incorporated by reference). Eventually the extracted collagen can be converted into a finely divided fibrous collagen by treating water-wet collagen with acetone to remove water, centrifuging to obtain the solid mass of collagen and deaggregating the collagen during drying. (See e.g., U.S. Pat. No. 4,148,664, herein incorporated by reference). The collagen preparation can then be brought back to a neutral pH and dried in the form of fibers. Completely transparent, physiological and hemocompatible gels, collagen films, and solutions can be prepared. These forms of collagen may then be used in the fabrication of contact lenses and implants.
One disadvantage of treatment with pepsin, is that the collagen preparation may be partially degraded (i.e., the extraction enzymes cleave the collagen molecule at the terminal non-helical regions, which contain the inter-collagenous cross-linkages). Indeed, it has been found that collagen extracted with pepsin results in preparations that are too weak for certain applications, especially those for which substantial mechanical handling of the collagen preparation is required.
Some acid treatments also have disadvantages. For example, the acid process described by Chvapil (M. Chvapil et al., Intl. Rev. Connective Tiss. Res., 6:1-55 [1979]) involves acid solubilization of bovine tendon collagen to produce a collagen suspension. This suspension is then either dialyzed or precipitated in saline, resulting in an amorphous precipitate containing non-fibrillary denatured collagen. Collagen prepared according to this method is generally not directly suitable for medical purposes, as it lacks tensile strength in moist media and has little resistance against enzymatic degradation when applied to living tissue. In addition, denatured collagen or collagen that has undergone treatment to reform the physical and biological characteristics to approximate collagen in vivo is often not satisfactory. It often lacks the mechanical properties required for wet dressings, as it lacks the in vivo organized structure (i.e.,collagen fibers are not present in this artificial collagen).
Thus, current methods for collagen preparation are unsatisfactory. Clearly, there is a need for the development of improved methods for the high volume production of high quality collagen suitable for use in medical treatment.
The present invention relates to methods for preparing collagen from humans and other animals. In particular, the present invention provides methods for the preparation of collagen suitable for biomedical applications.
The present invention provides numerous embodiments for the purification of collagen. It is particularly preferred that the collagen purified according to one embodiment of the present invention be type I collagen.
In one embodiment, the present invention provides a method for purifying collagen comprising: providing a sample comprising collagen, first and second proteolytic enzyme preparations, and a reducing agent; exposing the collagen sample to the first proteolytic enzyme preparation to produce a first collagen solution; exposing the first collagen solution to the reducing agent to produce a second collagen solution; exposing the second collagen solution to the second proteolytic enzyme preparation to produce purified collagen.
In one alternative embodiment, the method of the present invention further comprises the step of de-epithelializing the sample prior to exposing the sample to the first proteolytic enzyme preparation.
In a preferred embodiment, the first and/or second proteolytic enzyme preparation comprises an enzyme in the cysteine class. In a particularly preferred embodiment, the first and/or second proteolytic enzyme preparation comprises papain.
In an alternate embodiment of the method of the present invention, the reducing agent is selected from the group consisting of sodium sulfide, dithiothreitol, glutathionine, and sodium borohydride. In a preferred embodiment, the reducing agent comprises sodium borohydride.
In yet another embodiment, the method of the present invention comprises the further step of exposing the purified collagen to a delipidation agent to produce dilipidated collagen. In a preferred embodiment of this method, the delipidation agent comprises a mixture comprising chloroform and methanol.
In a particularly preferred embodiment, the method of the present invention further comprises the steps of compressing the delipidated collagen to produce compressed collagen; dehydrating the compressed collagen to produce dehydrated collagen; and dispersing and drying the dehydrating collagen to form collagen fibers. Thus, in this embodiment of the methods of the present invention, the collagen fibers are dried.
In another alternative embodiment, the method of the present invention comprises the step of exposing the delipidated collagen to a phosphorylating agent to produce phosphorylated collagen. In a preferred embodiment, the phosphorylation agent is selected from the group consisting of sodium trimetaphosphate, sodium hexametaphosphate, sodium ultraphosphate, sodium tetrametaphosphate, phosphoric anhydride, and phosphoryl trichloride. In a particularly preferred embodiment, the phosphorylation agent comprises sodium trimetaphosphate. In an alternatively preferred embodiment, the purified collagen comprises CollagenPRO(trademark).
In one embodiment, the present invention provides purified collagen purified by the steps of: providing a sample comprising collagen, first and second proteolytic enzyme preparations, and a reducing agent; exposing the collagen sample to the first proteolytic enzyme preparation to produce a first collagen solution; exposing the first collagen solution to the reducing agent to produce a second collagen solution; exposing the second collagen solution to the second proteolytic enzyme preparation to produce purified collagen.
It is further contemplated that the purified collagen be comprised of additional compounds, including but not limited to antimicrobials, antivirals, growth factors, anti-dehydration compounds, antiseptics, or other compounds suitable for biomedical and/or veterinary uses.
The present invention also provides an alternative embodiment comprising methods for production of biocompatible collagen, in which the method comprises: providing a sample comprising collagen, first and second proteolytic enzyme preparations, a reducing agent, a delipidation agent, and a phosphorylation agent; exposing the collagen sample to the first proteolytic enzyme preparation to produce a first collagen solution; exposing the first collagen solution to the reducing agent to produce a second collagen solution; exposing the second collagen solution to the second proteolytic enzyme preparation to produce to produce a proteolyzed collagen solution; exposing the proteolyzed collagen solution to the delipidation agent to produce delipidated collagen; and exposing the delipidated collagen to the phosphorylation agent to produce phosphorylated collagen.
In an alternative embodiment, the method of the present invention further comprises the step of de-epithelializing the sample prior to exposing the sample to the first proteolytic enzyme preparation.
In a preferred embodiment, the first and/or second proteolytic enzyme preparation of the method comprises an enzyme in the cysteine class. In a particularly preferred embodiment, the first and/or second proteolytic enzyme preparation comprises papain.
In an alternate embodiment of the method of the present invention, the reducing agent is selected from the group consisting of sodium sulfide, dithiothicitol, glutathionine, and sodium borohydride. In a preferred embodiment, the reducing agent comprises sodium borohydride.
In an alternative embodiment, the delipidationegent comprises a mixture of chloroform and methanol. In yet another embodiment, the phosphorylation agent is selected from the group consisting of sodium trimetaphosphate, sodium hexametaphosphate, sodium ultraphosphate, sodium tetrametaphosphate, phosphoric anhydride, and phosphoryl trichloride. In a particularly preferred embodiment, the phosphorylation agent comprises sodium trimetaphosphate.
In yet a further embodiment, the method of the present invention further comprises the steps of compressing the delipidated collagen to produce compressed collagen; dehydrating the compressed collagen to produce dehydrated collagen; and dispersing and drying the dehydrating collagen to form collagen fibers. Thus, in this embodiment of the methods of the present invention, the collagen fibers are dried. In a particularly preferred embodiment, the method further comprises the step of filter-sterilizing the delipidated collagen prior to exposing the delipidated collagen to the phosphorylation agent to produce phosphorylated collagen.
The present invention also provides purified such that the collagen is biocorripatible. In this embodiment, the biocompatible collagen is produced by the method of: providing a sample comprising collagen, first and second proteolytic enzyme preparations, a reducing agent, a delipidation agent, and a phosphorylation agent; exposing the collagen sample to the first proteolytic enzyme preparation to produce a first collagen solution; exposing the first collagen solution to the reducing agent to produce a second collagen solution; exposing the second collagen solution to the second proteolytic enzyme preparation to produce to produce a proteolyzed collagen solution; exposing the proteolyzed collagen solution to the delipidation agent to produce delipidated collagen; and exposing the delipidated collagen to the phosphorylation agent to produce phosphorylated collagen. In one preferred embodiment, the phosphorylated collagen comprises CollagenPRO(trademark).
In another preferred embodiment, the biocompatible collagen comprises a film or membrane, while in alternate preferred embodiments, the biocompatible collagen comprises a solid, while in other alternately preferred embodiments, the biocompatible collagen comprises a solution. In other embodiments, the biocompatible collagen is a dried film that is hydrated prior to its application.
It is further contemplated that the biocompatible collagen be comprised of additional compounds, including but not limited to antimicrobials, antivirals, growth factors, anti-dehydration compounds, antiseptics, or other compounds suitable for biomedical and/or veterinary uses.
The present invention also provides methods for achieving hemostasis comprising: providing purified and/or biocompatible collagen purified as described above, and a bleeding wound; and exposing the purified and/or biocompatible collagen to the bleeding wound.
The present invention also provides a method of transplantation comprising providing: purified and/or biocompatible collagen, and a transplantation site; and exposing the purfied and/or biocompatible collagen to the transplantation site.